SRM/MRM assay for the ephrin type-A receptor 2 protein

ABSTRACT

Specific peptides, and derived ionization characteristics of the peptides, from the Ephrin Type-A Receptor 2 (EPHA2) protein are provided that are particularly advantageous for quantifying the EPHA2 protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed and are selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from said biological sample using the Liquid Tissue™ reagents and protocol and the EPHA2 protein is quantitated in the Liquid Tissue™ sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of an EPHA2 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

This application is a continuation of International Application No.PCT/US12/58815, filed Oct. 4, 2012, which claims the benefit of U.S.Provisional Application No. 61/543,106, filed Oct. 4, 2011, entitled“SRM/MRM Assay for the Ephrin Type-A Receptor 2 Protein,” the contentsof each of which are hereby incorporated by referenced in theirentireties. This application also contains a sequence listing submittedelectronically via EFS-web, which serves as both the paper copy and thecomputer readable form (CRF) and consists of a file entitled“001152_8027_US01_SEQ_LISTING”, which was created on Mar. 25, 2014,which is 1,024 bytes in size, and which is also incorporated byreference in its entirety.

INTRODUCTION

Specific peptides derived from subsequences of the Ephrin Type-AReceptor 2 protein (referred to herein as EPHA2), are provided. Thepeptide sequence and fragmentation/transition ions for each peptide areparticularly useful in a mass spectrometry-based Selected ReactionMonitoring (SRM) assay, which can also be referred to as a MultipleReaction Monitoring (MRM) assay. Such assays are referred to herein asSRM/MRM. The use of peptides for quantitative SRM/MRM analysis of theEPHA2 protein is described.

This SRM/MRM assay can be used to measure relative or absolutequantitative levels of one or more of the specific peptides from theEPHA2 protein. This provides a means of measuring the amount of theEPHA2 protein in a given protein preparation obtained from a biologicalsample by mass spectrometry.

More specifically, the SRM/MRM assay can measure these peptides directlyin complex protein lysate samples prepared from cells procured frompatient tissue samples, such as formalin fixed cancer patient tissue.Methods of preparing protein samples from formalin-fixed tissue aredescribed in U.S. Pat. No. 7,473,532, the contents of which are herebyincorporated by reference in their entirety. The methods described inU.S. Pat. No. 7,473,532 may conveniently be carried out using LiquidTissue™ reagents and protocol available from OncoPlexDx (formerlyExpression Pathology Inc., Rockville, Md.).

The most widely and advantageously available form of tissues from cancerpatients tissue is formalin fixed, paraffin embedded tissue.Formaldehyde/formalin fixation of surgically removed tissue is by farand away the most common method of preserving cancer tissue samplesworldwide and is the accepted convention for standard pathologypractice. Aqueous solutions of formaldehyde are referred to as formalin.“100%” formalin consists of a saturated solution of formaldehyde (thisis about 40% by volume or 37% by mass) in water, with a small amount ofstabilizer, usually methanol to limit oxidation and degree ofpolymerization. The most common way in which tissue is preserved is tosoak whole tissue for extended periods of time (8 hours to 48 hours) inaqueous formaldehyde, commonly termed 10% neutral buffered formalin,followed by embedding the fixed whole tissue in paraffin wax for longterm storage at room temperature. Thus molecular analytical methods toanalyze formalin fixed cancer tissue will be the most accepted andheavily utilized methods for analysis of cancer patient tissue.

Results from the SRM/MRM assay can be used to correlate accurate andprecise quantitative levels of the EPHA2 protein within the specifictissue samples (e.g., one or more cancer tissue samples) of the patientor subject from whom the tissue (biological sample) was collected andpreserved. This not only provides diagnostic information about thecancer, but also permits a physician or other medical professional todetermine appropriate therapy for the patient. For example, such anassay can be designed to diagnose the stage or degree of a cancer anddetermine a therapeutic agent to which a patient is most likely torespond. Such an assay that provides diagnostically and therapeuticallyimportant information about levels of protein expression in a diseasedtissue or other patient sample is termed a companion diagnostic assay.

SUMMARY

The assays described herein measure relative or absolute levels ofspecific unmodified peptides from the EPHA2 protein and also can measureabsolute or relative levels of specific modified peptides from the EPHA2protein. Examples of modifications include phosphorylated amino acidresidues (e.g. phosphotyrosine, phosphoserine and phosphothreonine) andglycosylated amino acid residues (e.g. glycosylated asparagine residues)that are present on the peptides.

Relative quantitative levels of the EPHA2 protein are determined by theSRM/MRM methodology for example by comparing SRM/MRM signature peakareas (e.g., signature peak area or integrated fragment ion intensity)of an individual EPHA2 peptide in different samples. Alternatively, itis possible to compare multiple SRM/MRM signature peak areas formultiple EPHA2 signature peptides, where each peptide has its ownspecific SRM/MRM signature peak, to determine the relative EPHA2 proteincontent in one biological sample with the EPHA2 protein content in oneor more additional or different biological samples. In this way, theamount of a particular peptide, or peptides, from the EPHA2 protein, andtherefore the amount of the EPHA2 protein, is determined relative to thesame EPHA2 peptide, or peptides, across 2 or more biological samplesunder the same experimental conditions. In addition, relativequantitation can be determined for a given peptide, or peptides, fromthe EPHA2 protein within a single sample by comparing the signature peakarea for that peptide by SRM/MRM methodology to the signature peak areafor another and different peptide, or peptides, from a differentprotein, or proteins, within the same protein preparation from thebiological sample. In this way, the amount of a particular peptide fromthe EPHA2 protein, and therefore the amount of the EPHA2 protein, isdetermined relative one to another within the same sample. Theseapproaches generate quantitation of an individual peptide, or peptides,from the EPHA2 protein to the amount of another peptide, or peptides,between samples and within samples wherein the amounts as determined bypeak area are relative one to another, regardless of the absolute weightto volume or weight to weight amounts of the EPHA2 peptide in theprotein preparation from the biological sample. Relative quantitativedata about individual signature peak areas between different samples arenormalized to the amount of protein analyzed per sample. Relativequantitation can be performed across many peptides from multipleproteins and the EPHA2 protein simultaneously in a single sample and/oracross many samples to gain insight into relative protein amounts, onepeptide/protein with respect to other peptides/proteins.

Absolute quantitative levels of the EPHA2 protein are determined by, forexample, the SRM/MRM methodology whereby the SRM/MRM signature peak areaof an individual peptide from the EPHA2 protein in one biological sampleis compared to the SRM/MRM signature peak area of an exogenously added“spiked internal standard.” In one embodiment, the internal standard isa synthetic version of the same exact EPHA2 peptide that contains one ormore amino acid residues labeled with one or more heavy isotopes.Suitable isotope labeled internal standards are synthesized so that whenanalyzed by mass spectrometry each standard generates a predictable andconsistent SRM/MRM signature peak that is different and distinct fromthe native EPHA2 peptide signature peak and which can be used as acomparator peak. Thus when the internal standard is spiked in a knownamount into a protein preparation from a biological sample and analyzedby mass spectrometry, the SRM/MRM signature peak area of the nativepeptide from the sample can be compared to the SRM/MRM signature peakarea of the internal standard peptide. This numerical comparisonprovides either the absolute molarity and/or absolute weight of thenative peptide present in the original protein preparation from thebiological sample. Absolute quantitative data for fragment peptides aredisplayed according to the amount of protein analyzed per sample.Absolute quantitation can be performed across many peptides, and thusproteins, simultaneously in a single sample and/or across many samplesto gain insight into absolute protein amounts in individual biologicalsamples and in entire cohorts of individual samples.

The SRM/MRM assay method can be used to aid diagnosis of the stage ofcancer, for example, directly in patient-derived tissue, such asformalin fixed tissue, and to aid in determining which therapeutic agentwould be most advantageous for use in treating that patient. Cancertissue that is removed from a patient either through surgery, such asfor therapeutic removal of partial or entire tumors, or through biopsyprocedures conducted to determine the presence or absence of suspecteddisease, is analyzed to determine whether or not a specific protein, orproteins, and which forms of proteins, are present in that patienttissue. Moreover, the expression level of a protein, or multipleproteins, can be determined and compared to a “normal” or referencelevel found in healthy tissue. Normal or reference levels of proteinsfound in healthy tissue may be derived from, for example, the relevanttissues of one or more individuals that do not have cancer.Alternatively, normal or reference levels may be obtained forindividuals with cancer by analysis of relevant tissues not affected bythe cancer.

Assays of protein levels (e.g., EPHA2 levels) can also be used todiagnose the stage of cancer in a patient or subject diagnosed withcancer by employing the EPHA2 levels. Levels or amounts of proteins orpeptides can be defined as the quantity expressed in moles, mass orweight of a protein or peptide determined by the SRM/MRM assay. Thelevel or amount may be normalized to total the level or amount ofprotein or another component in the lysate analyzed (e.g., expressed inmicromoles/microgram of protein or micrograms/microgram of protein). Inaddition, the level or amount of a protein or peptide may be determinedon volume basis, expressed, for example, in micromolar ornanograms/microliter. The level or amount of protein or peptide asdetermined by the SRM/MRM assay can also be normalized to the number ofcells analyzed. Information regarding EPHA2 can thus be used to aid indetermining stage or grade of a cancer by correlating the level of theEPHA2 protein (or fragment peptides of the EPHA2 protein) with levelsobserved in normal tissues.

Once the stage and/or grade, and/or EPHA2 protein expressioncharacteristics of the cancer has been determined, that information canbe matched to a list of therapeutic agents (chemical and biological)developed to specifically treat cancer tissue that is characterized by,for example, abnormal expression of the protein or protein(s) (e.g.,EPHA2) that were assayed. Matching information from an EPHA2 proteinassay to a list of therapeutic agents that specifically targets, forexample, the EPHA2 protein or cells/tissue expressing the protein,defines what has been termed a personalized medicine approach totreating disease. The assay methods described herein form the foundationof a personalized medicine approach by using analysis of proteins fromthe patient's own tissue as a source for diagnostic and treatmentdecisions.

DETAILED DESCRIPTION

In principle, any predicted peptide derived from EPHA2 protein, preparedfor example by digesting with a protease of known specificity (e.g.trypsin), can be used as a surrogate reporter to determine the abundanceof EPHA2 protein in a sample using a mass spectrometry-based SRM/MRMassay. Similarly, any predicted peptide sequence containing an aminoacid residue at a site that is known to be potentially modified in EPHA2protein also can be used to assay the extent of modification of EPHA2protein in a sample.

EPHA2 fragment peptides may be generated by a variety of ways includingusing the Liquid Tissue™ protocol described, for example, in U.S. Pat.No. 7,473,532. The Liquid Tissue™ protocol and reagents produce peptidesamples suitable for mass spectroscopic analysis from formalin fixedparaffin embedded tissue by proteolytic digestion of the proteins in thetissue/biological sample. Suitable reagents and protocols also arecommercially available from OncoPlexDx (formerly Expression PathologyInc., Rockville, Md.).

In the Liquid Tissue™ protocol the tissue/biological sample is heated ina buffer for an extended period of time (e.g., from about 80° C. toabout 100° C. for a period of time from about 10 minutes to about 4hours) to reverse or release protein cross-linking. The buffer employedis a neutral buffer, (e.g., a Tris-based buffer, or a buffer containinga detergent). Following heat treatment the tissue/biological sample istreated with one or more proteases, including but not limited totrypsin, chymotrypsin, pepsin, and endoproteinase Lys-C for a timesufficient to disrupt the tissue and cellular structure of saidbiological sample and to liquefy the sample. Exemplary conditions forthe protease treatment are from about 30 minutes to about 24 hours at atemperature from about 37° C. to about 65° C.). Advantageously,endoproteases, and particularly combinations of two or threeendoproteases, used either simultaneously or sequentially, are employedto liquefy the sample. For example, suitable combinations of proteasescan include, but are not limited to, combinations of trypsin,endoproteinase Lys-C and chemotrypsin, such as trypsin andendoproteinase Lys-C. The result of the heating and proteolysis is aliquid, soluble, dilutable biomolecule lysate. Advantageously, thisliquid lysate is free of solid or particulate matter that can beseparated from the lysate by centrifugation.

Surprisingly, it was found that many potential peptide sequences fromthe EPHA2 protein are unsuitable or ineffective for use in massspectrometry-based SRM/MRM assays for reasons that are not immediatelyevident. As it was not possible to predict the most suitable peptidesfor MRM/SRM assay, it was necessary to experimentally identify modifiedand unmodified peptides in actual Liquid Tissue™ lysates to develop areliable and accurate SRM/MRM assay for the EPHA2 protein. While notwishing to be bound by any theory, it is believed that some peptidesmight, for example, be difficult to detect by mass spectrometry becausethey do not ionize well, or produce fragments that are not distinct fromthose generated from other proteins. Peptides may also fail to resolvewell in separation (e.g., liquid chromatography), or may adhere to glassor plastic ware, which leads to erroneous results in the EM/MRM assay.

EPHA2 peptides found in various embodiments of this disclosure (e.g.,Tables 1 and 2 below) were derived from the EPHA2 protein by proteasedigestion of all the proteins within a complex Liquid Tissue™ lysateprepared from cells procured from formalin fixed cancer tissue. Unlessnoted otherwise, in each instance the protease was trypsin. The LiquidTissue™ lysate was then analyzed by mass spectrometry to determine thosepeptides derived from the EPHA2 protein that are detected and analyzedby mass spectrometry. Identification of a specific preferred subset ofpeptides for mass-spectrometric analysis is based on; 1) experimentaldetermination of which peptide or peptides from a protein ionize in massspectrometry analyses of Liquid Tissue™ lysates, and 2) the ability ofthe peptide to survive the protocol and experimental conditions used inpreparing a Liquid Tissue™ lysate. This latter property extends not onlyto the amino acid sequence of the peptide but also to the ability of amodified amino acid residue within a peptide to survive in modified formduring the sample preparation.

Protein lysates from cells procured directly from formalin(formaldehyde) fixed tissue were prepared using the Liquid Tissue™reagents and protocol. This entails collecting cells into a sample tubevia tissue microdissection followed by heating the cells in the LiquidTissue™ buffer for an extended period of time. Once the formalin-inducedcross linking has been negatively affected, the tissue/cells are thendigested to completion in a predictable manner using a protease, such astrypsin. The skilled artisan will recognize that other proteases, and inparticular, endoproteases may be used in place of, or in addition to,trypsin. Each protein lysate was used to prepare a collection ofpeptides by digestion of intact polypeptides with the protease orprotease combination. Each Liquid Tissue™ lysate was analyzed (e.g., byion trap mass spectrometry) to perform multiple global proteomic surveysof the peptides where the data was presented as identification of asmany peptides as could be identified by mass spectrometry from allcellular proteins present in each protein lysate. An ion trap massspectrometer or another form of a mass spectrometer that is capable ofperforming global profiling for identification of as many peptides aspossible from a single complex protein/peptide lysate may be employed.Ion trap mass spectrometers may, however, be the best type of massspectrometer presently available for conducting global profiling ofpeptides. Although SRM/MRM assay can be developed and performed on anytype of mass spectrometer, including a MALDI, ion trap, or triplequadrupole, an advantageous instrument platform for SRM/MRM assay isoften considered to be a triple quadrupole instrument platform.

Once as many peptides as possible were identified in a single massspectrometric analysis of a single lysate under the conditions employed,then that list of peptides was collated and used to determine theproteins that were detected in that lysate. That process was repeatedfor multiple Liquid Tissue™ lysates, and the very large list of peptideswas collated into a single dataset. The resulting dataset represents thepeptides that can be detected in the type of biological sample that wasanalyzed (after protease digestion), and specifically in a LiquidTissue™ lysate of the biological sample, and thus includes the peptidesfor specific proteins, such as for example the EPHA2 protein.

In one embodiment, the EPHA2 tryptic peptides identified as useful inthe determination of absolute or relative amounts of the EPHA2 receptorinclude either one or both of the peptides of SEQ ID NO:1 and SEQ IDNO:2, the sequences of each of which are shown in Table 1. Each of thosepeptides was detected by mass spectrometry in Liquid Tissue™ lysatesprepared from formalin fixed, paraffin embedded tissue. Thus, either oneof the peptides individually or both peptides in combination found inTable 1 are candidates for use in quantitative SRM/MRM assay for theEPHA2 protein in human biological samples, including directly informalin fixed patient tissue. Table 2 shows additional informationregarding the peptides shown in Table 1.

TABLE 1 SEQ ID Peptide sequence SEQ ID NO: 1 TASVSINQTEPPK SEQ ID NO: 2IDTIAPDEITVSSDFEAR

TABLE 2 Mono Precursor Precursor Transition SEQ ID Peptide sequenceIsotopic Charge m/z m/z Ion Type SEQ ID NO: 1 TASVSINQTEPPK 1370.7 2686.359 813.41 y7 2 686.359 926.494 y8 2 686.359 1013.526 y9 2 690.366821.424 y7 2 690.366 934.508 y8 2 690.366 1021.54 y9 SEQ ID NO: 2IDTIAPDEITVSSDFEAR 1977.95 2 989.984 811.358 y7 2 989.984 910.426 y8 2989.984 1011.474 y9 2 989.984 1124.558 y10 2 989.984 1253.6 y11 2989.984 1368.627 y12 2 989.984 1465.68 y13

The EPHA2 tryptic peptides listed in Table 1 include those detected frommultiple Liquid Tissue™ lysates of multiple different formalin fixedtissues of different human organs including prostate, colon, and breast.Each of those peptides is useful for quantitative SRM/MRM assay of theEPHA2 protein in formalin fixed tissue. Further data analysis of theseexperiments indicated no preference is observed for any specificpeptides from any specific organ site. Thus, each of these peptides isbelieved to be suitable for conducting SRM/MRM assays of the EPHA2protein on a Liquid Tissue™ lysate from any formalin fixed tissueoriginating from any biological sample or from any organ site in thebody.

In one embodiment the peptides in Table 1, or both of those peptides areassayed by methods that do not rely upon mass spectroscopy, including,but not limited to, immunological methods (e.g., Western blotting orELISA). Regardless of how information directed to the amount of thepeptide(s) (absolute or relative) is obtained, the information may beemployed in any of the methods described herein, including indicating(diagnosing) the presence of cancer in a subject, determining thestage/grade/status of the cancer, providing a prognosis, or determiningthe therapeutics or treatment regimen for a subject/patient.

Embodiments of the present disclosure include compositions comprisingeither peptide individually or both of the peptides in Table 1, asfurther characterized in Table 2. In specific embodiments, thecompositions comprise either peptide individually or both peptides incombination where one or both of the peptides are isotopically labeled.Each of the peptides may be labeled with one or more isotopes selectedindependently from the group consisting of: ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²Hor combinations thereof. Compositions comprising peptides from the EPHA2protein, whether isotope labeled or not, do not need to contain all ofthe peptides from that protein (e.g., a complete set of trypticpeptides). In some embodiments the compositions do not contain bothpeptides in combination from EPHA2, and particularly peptides appearingin Table 1 or Table 2. Compositions comprising peptides may be in theform of dried or lyophized materials, liquid (e.g., aqueous) solutionsor suspensions, arrays, or blots.

One consideration for conducting an SRM/MRM assay is the type ofinstrument that may be employed in the analysis of the peptides.Although SRM/MRM assays can be developed and performed on any type ofmass spectrometer, including a MALDI, ion trap, or triple quadrupole,the most advantageous instrument platform for SRM/MRM assay is oftenconsidered to be a triple quadrupole instrument platform. That type of amass spectrometer may be considered to be the most suitable instrumentfor analyzing a single isolated target peptide within a very complexprotein lysate that may consist of hundreds of thousands to millions ofindividual peptides from all the proteins contained within a cell.

In order to most efficiently implement SRM/MRM assay for each peptidederived from the EPHA2 protein it is desirable to utilize information inaddition to the peptide sequence in the analysis. That additionalinformation may be used in directing and instructing the massspectrometer (e.g. a triple quadrupole mass spectrometer) to perform thecorrect and focused analysis of specific targeted peptide(s) such thatthe assay may be effectively performed.

The additional information about target peptides in general, and aboutspecific EPHA2 peptides, may include one or more of the mono isotopicmass of the peptide, its precursor charge state, the precursor m/zvalue, the m/z transition ions, and the ion type of each transition ion.Additional peptide information that may be used to develop an SRM/MRMassay for the EPHA2 protein is shown in Table 2 for the two (2) EPHA2peptides from the list in Table 1.

The method described below was used to: 1) identify candidate peptidesfrom the EPHA2 protein that can be used for a mass spectrometry-basedSRM/MRM assay for the EPHA2 protein, 2) develop individual SRM/MRMassay, or assays, for target peptides from the EPHA2 protein, and 3)apply quantitative assays to cancer diagnosis and/or choice of optimaltherapy.

Assay Method

1. Identification of SRM/MRM Candidate Fragment Peptides for the EPHA2Protein

-   -   a. Prepare a Liquid Tissue™ protein lysate from a formalin fixed        biological sample using a protease or proteases, (that may or        may not include trypsin), to digest proteins    -   b. Analyze all protein fragments in the Liquid Tissue™ lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the EPHA2 protein, where individual fragment        peptides do not contain any peptide modifications such as        phosphorylations or glycosylations    -   c. Analyze all protein fragments in the Liquid Tissue™ lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the EPHA2 protein that carry peptide modifications        such as for example phosphorylated or glycosylated residues    -   d. All peptides generated by a specific digestion method from        the entire, full length EPHA2 protein potentially can be        measured, but preferred peptides used for development of the        SRM/MRM assay are those that are identified by mass spectrometry        directly in a complex Liquid Tissue™ protein lysate prepared        from a formalin fixed biological sample    -   e. Peptides that are specifically modified (phosphorylated,        glycosylated, etc.) in patient tissue and which ionize, and thus        detected, in a mass spectrometer when analyzing a Liquid Tissue™        lysate from a formalin fixed biological sample are identified as        candidate peptides for assaying peptide modifications of the        EPHA2 protein        2. Mass Spectrometry Assay for Fragment Peptides from EPHA2        Protein    -   a. SRM/MRM assay on a triple quadrupole mass spectrometer for        individual fragment peptides identified in a Liquid Tissue™        lysate is applied to peptides from the EPHA2 protein        -   i. Determine optimal retention time for a fragment peptide            for optimal chromatography conditions including but not            limited to gel electrophoresis, liquid chromatography,            capillary electrophoresis, nano-reversed phase liquid            chromatography, high performance liquid chromatography, or            reverse phase high performance liquid chromatography        -   ii. Determine the mono isotopic mass of the peptide, the            precursor charge state for each peptide, the precursor m/z            value for each peptide, the m/z transition ions for each            peptide, and the ion type of each transition ion for each            fragment peptide in order to develop an SRM/MRM assay for            each peptide.        -   iii. SRM/MRM assay can then be conducted using the            information from (i) and (ii) on a triple quadrupole mass            spectrometer where each peptide has a characteristic and            unique SRM/MRM signature peak that precisely defines the            unique SRM/MRM assay as performed on a triple quadrupole            mass spectrometer    -   b. Perform SRM/MRM analysis so that the amount of the fragment        peptide of the EPHA2 protein that is detected, as a function of        the unique SRM/MRM signature peak area from an SRM/MRM mass        spectrometry analysis, can indicate both the relative and        absolute amount of the protein in a particular protein lysate.        -   i. Relative quantitation may be achieved by:            -   1. Determining increased or decreased presence of the                EPHA2 protein by comparing the SRM/MRM signature peak                area from a given EPHA2 peptide detected in a Liquid                Tissue™ lysate from one formalin fixed biological sample                to the same SRM/MRM signature peak area of the same                EPHA2 fragment peptide in at least a second, third,                fourth or more Liquid Tissue™ lysates from least a                second, third, fourth or more formalin fixed biological                samples            -   2. Determining increased or decreased presence of the                EPHA2 protein by comparing the SRM/MRM signature peak                area from a given EPHA2 peptide detected in a Liquid                Tissue™ lysate from one formalin fixed biological sample                to SRM/MRM signature peak areas developed from fragment                peptides from other proteins, in other samples derived                from different and separate biological sources, where                the SRM/MRM signature peak area comparison between the 2                samples for a peptide fragment are normalized to amount                of protein analyzed in each sample.            -   3. Determining increased or decreased presence of the                EPHA2 protein by comparing the SRM/MRM signature peak                area for a given EPHA2 peptide to the SRM/MRM signature                peak areas from other fragment peptides derived from                different proteins within the same Liquid Tissue™ lysate                from the formalin fixed biological sample in order to                normalize changing levels of EPHA2 protein to levels of                other proteins that do not change their levels of                expression under various cellular conditions.            -   4. These assays can be applied to both unmodified                fragment peptides and for modified fragment peptides of                the EPHA2 protein, where the modifications include but                are not limited to phosphorylation and/or glycosylation,                and where the relative levels of modified peptides are                determined in the same manner as determining relative                amounts of unmodified peptides.        -   ii. Absolute quantitation of a given peptide may be achieved            by comparing the SRM/MRM signature peak area for a given            fragment peptide from the EPHA2 protein in an individual            biological sample to the SRM/MRM signature peak area of an            internal fragment peptide standard spiked into the protein            lysate from the biological sample            -   1. The internal standard is a labeled synthetic version                of the fragment peptide from the EPHA2 protein that is                being interrogated. This standard is spiked into a                sample in known amounts, and the SRM/MRM signature peak                area can be determined for both the internal fragment                peptide standard and the native fragment peptide in the                biological sample separately, followed by comparison of                both peak areas            -   2. This can be applied to unmodified fragment peptides                and modified fragment peptides, where the modifications                include but are not limited to phosphorylation and/or                glycosylation, and where the absolute levels of modified                peptides can be determined in the same manner as                determining absolute levels of unmodified peptides.                3. Apply Fragment Peptide Quantitation to Cancer                Diagnosis and Treatment    -   a. Perform relative and/or absolute quantitation of fragment        peptide levels of the EPHA2 protein and demonstrate that the        previously-determined association, as well understood in the        field of cancer, of EPHA2 protein expression to the        stage/grade/status of cancer in patient tumor tissue is        confirmed    -   b. Perform relative and/or absolute quantitation of fragment        peptide levels of the EPHA2 protein and demonstrate correlation        with clinical outcomes from different treatment strategies,        wherein this correlation has already been demonstrated in the        field or can be demonstrated in the future through correlation        studies across cohorts of patients and tissue from those        patients. Once either previously established correlations or        correlations derived in the future are confirmed by this assay        then the assay method can be used to determine optimal treatment        strategy

Assessment of EPHA2 protein levels in tissues based on analysis offormalin fixed patient-derived tissue can provide diagnostic,prognostic, and therapeutically-relevant information about eachparticular patient. In one embodiment, this disclosure describes amethod for measuring the level of the EPHA2 protein in a biologicalsample, comprising detecting and/or quantifying the amount of one ormore modified or unmodified EPHA2 fragment peptides in a protein digestprepared from said biological sample using mass spectrometry; andcalculating the level of modified or unmodified EPHA2 protein in saidsample; and wherein said level is a relative level or an absolute level.In a related embodiment, quantifying one or more EPHA2 fragment peptidescomprises determining the amount of the each of the EPHA2 fragmentpeptides in a biological sample by comparison to a known amount of anadded internal standard peptide of known amount, where each of the EPHA2fragment peptides in the biological sample is compared to an internalstandard peptide having the same amino acid sequence. In someembodiments the internal standard is an isotopically labeled internalstandard peptide comprises one or more heavy stable isotopes selectedfrom ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinations thereof.

The method for measuring the level of the EPHA2 protein in a biologicalsample described herein (or fragment peptides as surrogates thereof) maybe used as a diagnostic indicator of cancer in a patient or subject. Inone embodiment, the results from measurements of the level of the EPHA2protein may be employed to determine the diagnostic stage/grade/statusof a cancer by correlating (e.g., comparing) the level of EPHA2 proteinfound in a tissue with the level of that protein found in normal and/orcancerous or precancerous tissues.

Embodiments

1. A method for measuring the level of the Ephrin Type-A Receptor 2(EPHA2) protein in a biological sample, comprising detecting and/orquantifying the amount of one or more modified or unmodified EPHA2fragment peptides in a protein digest prepared from said biologicalsample using mass spectrometry; and calculating the level of modified orunmodified EPHA2 protein in said sample; and wherein said amount is arelative amount or an absolute amount.2. The method of embodiment 1, further comprising the step offractionating said protein digest prior to detecting and/or quantifyingthe amount of one or more modified or unmodified EPHA2 fragmentpeptides.3. The method of embodiment 2, wherein said fractionating step isselected from the group consisting of gel electrophoresis, liquidchromatography, capillary electrophoresis, nano-reversed phase liquidchromatography, high performance liquid chromatography, or reverse phasehigh performance liquid chromatography.4. The method of any of embodiments 1-3, wherein said protein digest ofsaid biological sample is prepared by the Liquid Tissue™ protocol.5. The method of any of embodiments 1-3, wherein said protein digestcomprises a protease digest.6. The method of embodiment 5, wherein said protein digest comprises atrypsin digest.7. The method of any of embodiments 1-6, wherein said mass spectrometrycomprises tandem mass spectrometry, ion trap mass spectrometry, triplequadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI massspectrometry, and/or time of flight mass spectrometry.8. The method of embodiment 7, wherein the mode of mass spectrometryused is Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring(MRM), and/or multiple Selected Reaction Monitoring (mSRM), or anycombination thereof.9. The method of any of embodiments 1 to 8, wherein the EPHA2 fragmentpeptide comprises an amino acid sequence as set forth as SEQ ID NO:1 andSEQ ID NO:2.10. The method of any of embodiments 0-9, wherein the biological sampleis a blood sample, a urine sample, a serum sample, an ascites sample, asputum sample, lymphatic fluid, a saliva sample, a cell, or a solidtissue.11. The method of embodiment 10, wherein the tissue is formalin fixedtissue.12. The method of embodiment 10 or 11, wherein the tissue is paraffinembedded tissue.13. The method of embodiment 10, wherein the tissue is obtained from atumor.14. The method of embodiment 13, wherein the tumor is a primary tumor.15. The method of embodiment 13, wherein the tumor is a secondary tumor.16. The method of any of embodiments 0 to 15, further comprisingquantifying a modified or unmodified EPHA2 fragment peptide.17. The method of embodiment 16, wherein quantifying the EPHA2 fragmentpeptide comprises comparing an amount of one or more EPHA2 fragmentpeptides comprising an amino acid sequence of about 8 to about 45 aminoacid residues of EPHA2 as shown in SEQ ID NO:1 or SEQ ID NO:2 in onebiological sample to the amount of the same EPHA2 fragment peptide in adifferent and separate biological sample.18. The method of embodiment 17, wherein quantifying one or more EPHA2fragment peptides comprises determining the amount of the each of theEPHA2 fragment peptides in a biological sample by comparison to an addedinternal standard peptide of known amount, wherein each of the EPHA2fragment peptides in the biological sample is compared to an internalstandard peptide having the same amino acid sequence.19. The method of embodiment 18, wherein the internal standard peptideis an isotopically labeled peptide.20. The method of embodiment 19, wherein the isotopically labeledinternal standard peptide comprises one or more heavy stable isotopesselected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinations thereof.21. The method of any of embodiments 1 to 20, wherein detecting and/orquantifying the amount of one or more modified or unmodified EPHA2fragment peptides in the protein digest indicates the presence ofmodified or unmodified EPHA2 protein and an association with cancer inthe subject.22. The method of embodiment 21, further comprising correlating theresults of said detecting and/or quantifying the amount of one or moremodified or unmodified EPHA2 fragment peptides, or the amount of saidEPHA2 protein to the diagnostic stage/grade/status of the cancer.23. The method of embodiment 22, wherein correlating the results of saiddetecting and/or quantifying the amount of one or more modified orunmodified EPHA2 fragment peptides, or the amount of said EPHA2 proteinto the diagnostic stage/grade/status of the cancer is combined withdetecting and/or quantifying the amount of other proteins or peptidesfrom other proteins in a multiplex format to provide additionalinformation about the diagnostic stage/grade/status of the cancer.24. The method of any one of embodiments 1-23, further comprisingselecting for the subject from which said biological sample was obtaineda treatment based on the presence, absence, or amount of one or moreEPHA2 fragment peptides or the amount of EPHA2 protein.25. The method of any one of embodiments 1-24, further comprisingadministering to the patient from which said biological sample wasobtained a therapeutically effective amount of a therapeutic agent,wherein the therapeutic agent and/or amount of the therapeutic agentadministered is based upon amount of one or more modified or unmodifiedEPHA2 fragment peptides or the amount of EPHA2 protein.26. The method of embodiments 24 and 25, wherein the treatment or thetherapeutic agent is directed to cancer cells expressing the EPHA2protein.27. The method of embodiments 1 to 26, wherein the biological sample isformalin fixed tumor tissue that has been processed for quantifying theamount of one or more modified or unmodified EPHA2 fragment peptidesemploying the Liquid Tissue™ protocol and reagents.28. The method of any of embodiments 1-27, wherein said one or moremodified or unmodified EPHA2 fragment peptides is one or more of thepeptides in Table 1.29. The method of any of embodiments 1-28, comprising quantifying theamount of the peptides in Table 2.30. A composition comprising either one individually or both incombination of the peptides in Table 1 or antibodies thereto.

The invention claimed is:
 1. A method for measuring the level of theEphrin Type-A Receptor 2 (EPHA2) protein in a human biological sample offormalin-fixed tumor tissue, comprising detecting and quantifying theamount of an unmodified EPHA2 fragment peptide in a protease digestprepared from said biological sample using mass spectrometry; andcalculating the level of EPHA2 protein in said sample; wherein the EPHA2fragment peptide consists of SEQ ID NO:1, and wherein said amount is anabsolute amount.
 2. The method of claim 1, further comprising the stepof fractionating said protease digest prior to detecting and/orquantifying the amount of said EPHA2 fragment peptide.
 3. The method ofclaim 2, wherein said fractionating step is selected from the groupconsisting of gel electrophoresis, liquid chromatography, capillaryelectrophoresis, nano-reversed phase liquid chromatography, highperformance liquid chromatography, or reverse phase high performanceliquid chromatography.
 4. The method of claim 1, wherein said proteasedigest comprises a trypsin digest.
 5. The method of claim 1, whereinsaid mass spectrometry comprises tandem mass spectrometry, ion trap massspectrometry, triple quadrupole mass spectrometry, MALDI-TOF massspectrometry, MALDI mass spectrometry, and/or time of flight massspectrometry.
 6. The method of claim 5, wherein the mode of massspectrometry used is Selected Reaction Monitoring (SRM), MultipleReaction Monitoring (MRM), and/or multiple Selected Reaction Monitoring(mSRM), or any combination thereof.
 7. The method of claim 1, whereinthe tissue is paraffin embedded tissue.
 8. The method of claim 1,wherein quantifying said EPHA2 fragment peptide comprises determiningthe amount of said EPHA2 fragment peptide in a biological sample bycomparison to an added internal standard peptide of known amount,wherein said EPHA2 fragment peptide in the biological sample is comparedto an internal isotopically labeled standard peptide having the sameamino acid sequence.
 9. The method of claim 8, wherein the isotopicallylabeled internal standard peptide comprises one or more heavy stableisotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinationsthereof.
 10. The method of claim 1, wherein said tumor is a prostatetumor.
 11. The method of claim 1, wherein said tumor is a colon tumor.